Lassus was born in Mons in the County of Hainaut (modern day Belgium). Information about his early years is scanty, although some uncorroborated stories have survived, the most famous of which is that he was kidnapped three times because of the singular beauty of his singing voice. At the age of twelve, he left the Low Countries with Ferrante Gonzaga and went to Mantua, Sicily, and later Milan (from 1547 to 1549). While in Milan, he made the acquaintance of the madrigalist Spirito l'Hoste da Reggio, a formative influence on his early musical style.

He then worked as a singer and a composer for Costantino Castrioto in Naples in the early 1550s, and his first works are presumed to date from this time. Next he moved to Rome, where he worked for Cosimo I de' Medici, Grand Duke of Tuscany, who maintained a household there; and in 1553, he became maestro di cappella of the Basilica di San Giovanni in Laterano, the ecumenical mother church of Rome and a spectacularly prestigious post indeed for a man only twenty-one years old. However, he stayed there only for a year (Palestrina would assume this post a year later, in 1555).

No solid evidence survives for his whereabouts in 1554, but there are contemporary claims that he traveled in France and England. In 1555 he returned to the Low Countries and had his early works published in Antwerp (1555-1556). In 1556 he joined the court of Albrecht V, Duke of Bavaria, who was consciously attempting to create a musical establishment on a par with the major courts in Italy. Lassus was one of several Netherlanders to work there, and by far the most famous. He evidently was happy in Munich and decided to settle there. In 1558 he married Regina WÃ¤ckinger, the daughter of a maid of honor of the Duchess; they had two sons, both of whom became composers. By 1563 Lassus had been appointed maestro di cappella, succeeding Ludwig Daser in the post. Lassus remained in the service of Albrecht V and his heir, Wilhelm V, for the rest of his life.

By the 1560s Lassus had become quite famous, and composers began to go to Munich to study with him. Andrea Gabrieli went there in 1562, and possibly remained in the chapel for a year; Giovanni Gabrieli also possibly studied with him in the 1570s. His renown had spread outside of strictly musical circles, for in 1570 Emperor Maximilian II conferred nobility upon him, a rare circumstance for a composer; Pope Gregory XIII knighted him; and in 1571, and again in 1573, the king of France, Charles IX, invited him to visit. Some of these kings and aristocrats attempted to woo him away from Munich with more attractive offers, but Lassus was evidently more interested in the stability of his position, and the splendid performance opportunities of Albrecht's court, than in financial gain. "I do not want to leave my house, my garden, and the other good things in Munich," he wrote to the Duke of Saxony in 1580, upon receiving an offer for a position in Dresden.

In the late 1570s and 1580s Lassus made several visits to Italy, where he encountered the most modern styles and trends. In Ferrara, the center of avant-garde activity, he doubtless heard the madrigals being composed for the d'Este court; however his own style remained conservative and became simpler and more refined as he aged. In the 1590s his health began to decline, and he went to a doctor named Thomas Mermann for treatment of what was called "melancholia hypocondriaca", but he was still able to compose as well as travel occasionally. His final work was often considered one of his best pieces: an exquisite set of twenty-one madrigali spirituali known as the Lagrime di San Pietro ("Tears of St. Peter"), which he dedicated to Pope Clement VIII, and which was published posthumously in 1595. Lassus died in Munich on 14 June 1594, the same day that his employer decided to dismiss him for economic reasons; he never saw the letter.

One of the most prolific, versatile, and universal composers of the late Renaissance, Lassus wrote over 2,000 works in all Latin, French, Italian and German vocal genres known in his time. These include 530 motets, 175 Italian madrigals and villanellas, 150 French chansons, and 90 German lieder. No strictly instrumental music by Lassus is known to survive, or ever to have existed: an interesting omission for a composer otherwise so wide-ranging and prolific, during an age when instrumental music was becoming an ever-more prominent means of expression, all over Europe.